Abstract
Cis-3-Hydroxypipecolic acid (cis-3-HyPip) is an important intermediate for the synthesis of GE81112 tetrapeptides, a small family of unusual nonribosomal peptide congeners with potent inhibitory activity against prokaryotic translation initiation. In this study, we constructed a microbial cell factory that can convert L-lysine into cis-3-hydroxypipecolic acid (cis-3-HyPip). Lysine cyclodeaminase SpLCD and Fe(II)/α-ketoglutarate (2-OG)-based oxygenase GetF were co-expressed in Escherichia coli. Plasmids with different copy numbers were used to balance the expression of these two enzymes, and the cell with the most appropriate balance of this kind for carrying plasmid pET-duet-getf-splcd was obtained. After determining the temperature (30 °C), pH (7.0), cell biomass, substrate concentration, Fe2+ concentration (10 mM), L-ascorbate concentration (10 mM), and TritonX-100 concentration (0.1% w/v) that were optimal for whole-cell catalysis, the yield of cis-3-HyPip reached as high as 25 mM (3.63 g/L).
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